Calorie restriction is one of the most widely studied interventions known to extend lifespan and/or healthspan in species as diverse as yeast and humans. Yeast has proven to be a fantastic model organism for the discovery of genes that influence human aging and age-related disease, due to the ease of study and the conservation of nutrient sensing and signaling pathways that underlie many aging processes. Initial studies in yeast pointed to Sir2, a histone deacetylase, as being responsible for mediating the life-extending properties of calorie restriction, while later studies called this research into question Confusion seems to relate to minor differences in strain background and media composition as well as more significant problems with the way calorie restriction is defined in yeast. Under a new yeast calorie restriction paradigm I developed, experiments point to significant Sir2-dependent and Sir2-independent pathways regulating chronological lifespan (the time a non-dividing cell remains viable in a stationary phase culture) in response to changing glucose concentrations. The major goal of this proposal is to investigate and define these pathways. My central hypotheses are that the Sirtuin-dependent and Sirtuin-independent pathways mediating yeast longevity will continue to be easier to elucidate under this experimental paradigm, that genes involved in traditional caloric restriction-mediated longevity operate across a much broader range of caloric conditions, and that Sir2 is likely affecting chronological lifespan through one or more of several known Sirtuin-linked longevity pathways in response to changes in sugar concentration. In Aim 1, both candidate and unbiased genetic approaches will be used to identify genes involved in the Sir2-independent glucose-regulated longevity pathway. Genes implicated previously in calorie restriction-mediated chronological longevity will be assayed for chronological lifespan in both high and low glucose conditions, and a pooled culture of the yeast knockout collection will be assayed for chronological longevity with short- and long-lived mutants identified by sequencing counts of unique DNA barcodes associated with each mutant. Mutants that fail to exhibit lifespan extension under calorie restriction conditions will be investigated further. In Aim 2, candidate and unbiased approaches will be used to identify genes involved in the Sir2-dependent pathway. Mutants that are defective in known or suspected Sir2- interacting pathways will be assayed for chronological lifespan in both caloric conditions, looking for epistatic interactions with sir2 . Additionally, deacetylation targets of Sir2 will be identifid using a process that biotinylates lysine residues specifically deacetylated by Sir2, allowing their precipitation and identification by tandem mass spectroscopy. Peptides subject to differential deacetylation under high and low calorie conditions will be investigated further for a role in chronological lifespan regulation. This work will identify new genes relevant to yeast chronological aging and calorie restriction-mediated longevity, and finally resolve the role of Sir in altering chronological lifespan in response to changing caloric conditions of growth media.